Immunoglobulin frameworks which demonstrate enhanced stability in the intracellular environment and methods of identifying same

ABSTRACT

Compositions are provided, which can be used as frameworks for the creation of very stable and soluble single-chain Fv antibody fragments. These frameworks have been selected for intracellular performance and are thus ideally suited for the creation of scFv antibody fragments or scFv antibody libraries for applications where stability and solubility are limiting factors for the performance of antibody fragments, such as in the reducing environment of a cell. Such frameworks can also be used to identify highly conserved residues and consensus sequences which demonstrate enhanced solubility and stability.

RELATED APPLICATIONS

This application is the U.S. National Phase Application pursuant to 35U.S.C. §371 of International Patent Application Serial No.PCT/EP03/05324 filed May 21, 2003, claiming benefit of priority of U.S.Provisional Application Ser. No. 60/382,649 filed May 22, 2002 and U.S.Provisional Application Ser. No. 60/438,256 filed Jan. 3, 2003. Theentire disclosure of each of the foregoing applications is incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to protein chemistry, molecular biology, andimmunology.

BACKGROUND OF THE RELATED ART

Antibodies can recognize and target almost any molecule with highspecificity and affinity. This characteristic has been exploited to turnthese natural proteins into powerful tools for diagnostic andtherapeutic applications. Advances in recombinant DNA technology havefacilitated the manipulation, cloning, and expression of antibody genesin a wide variety of non-lymphoid cells (Skerra, 1988; Martineau, 1998;Verma, 1998). A number of different antibody fragments have beenconstructed to best suit the various applications. The smallest entitythat retains the full antigen-binding capacity of the whole parentalimmunoglobulin is the single-chain Fv fragment (scFv) (Bird, 1988). Thisantibody fragment comprises the variable regions of the heavy and thelight chains linked by a flexible peptide-linker, which allows theexpression of the protein from a single gene.

Antibody fragments have several important advantages in comparison tothe entire immunoglobulin molecule. Due to their smaller size, theexpression is facilitated and the yield is enhanced in a variety ofexpression host cells, such as E. coli cells (Plückthun, 1996).Moreover, antibody fragments allow improved tumour penetration in invivo applications (Yokota, 1992) and they can be linked covalently tovarious effector molecules for therapeutic approaches.

Naturally occurring antibodies, which are secreted by plasma cells, haveevolved to function in an extracellular, oxidizing environment. Toobtain their functional, folded structure, they generally require theformation of disulfide-bridges within the separate domains, which arecrucial for the stability of the immunoglobulin fold. In contrast tofull-length antibodies, scFv or Fab antibody fragments can, inprinciple, be functionally expressed in a reducing environment insideany cell and directed to any compartment to target intracellularproteins and thus evoke specific biological effects (Biocca, 1991).Indeed, some intracellular single chain antibody fragments, which arecalled intrabodies, have been applied successfully to modulate thefunction of intracellular target proteins in different biologicalsystems. Thus, resistance against viral infections has been demonstratedin plant biotechnology (Tavladoraki, 1993; Benvenuto, 1995), binding ofintrabodies to HIV proteins has been shown (Rondon, 1997), and bindingto oncogene products (Biocca, 1993; Cochet, 1998; Lener, 2000) has beendescribed. Moreover, intracellular antibodies promise to be a valuabletool in characterizing the function of a vast number of genes nowidentified through the sequencing of the human genome (Richardson, 1995;Marasco, 1997). For example, they can be used in a functional genomicsapproach to block or modulate the activity of newly identified proteins,thereby contributing to the understanding of their functions. Finally,intrabodies have potential diagnostic and therapeutic applications, forexample in gene therapy settings.

Despite these great prospects, the generation of functional intrabodiesis still limited by their instability and insolubility or propensity toaggregate. The reducing environment of the cytoplasm prevents theformation of the conserved intrachain disulfide bridges, thus renderinga high percentage of antibody fragments unstable and, as a consequence,non-functional inside the cell (Biocca, 1995; Proba, 1997). Stabilityand solubility of antibody fragments therefore represents a majorobstacle for the application of intrabodies as potential modulators ofprotein function in vivo. So far, no predictions can be made about thesequence requirements that render an antibody fragment functional in anintracellular environment.

There is, therefore, a need for antibody fragments which perform well ina broad range of different cell types and can thus be used as frameworksfor diverse binding specificities. Such frameworks can be used toconstruct libraries for intracellular screening or can serve as anacceptor for the binding portions of an existing antibody.

Besides being uniquely suited for intracellular applications, suchantibody fragments or whole antibodies based on very stable variabledomain frameworks also have a distinct advantage over other antibodiesin numerous extracellular and in vitro applications. When suchframeworks are produced in an oxidizing environment, theirdisulfide-bridges can be formed, further enhancing their stability andmaking them highly resistant towards aggregation and proteasedegradation. The in vivo half-life (and thus the resistance towardsaggregation and degradation by serum proteases) is, besides affinity andspecificity, the single-most important factor for the success ofantibodies in therapeutic or diagnostic applications (Willuda, 1999).The half-life of antibody fragments can further be increased through thecovalent attachment of polymer molecules such as poly-ethylene glycol(PEG) (Weir, 2002). Stable molecules of this type represent asignificant advance in the use of antibodies, especially, but notexclusively, when the Fc functionality is not desired.

The great practical importance of antibody-fragment libraries hasmotivated research in this area. Winter (EP 0368684) has provided theinitial cloning and expression of antibody variable region genes.Starting from these genes he has created large antibody libraries havinghigh diversity in both the complementary determining regions (CDRs) aswell as in the framework regions. Winter does not disclose, however, theusefulness of different frameworks for library construction.

The teaching of Plückthun (EP 0859841), on the other hand, has tried toimprove the library design by limiting the frameworks to a definednumber of synthetic consensus sequences. Protein engineering effortsinvolving introduction of a large amount of rationally designedmutations have previously suggested mutations towards the respectiveconsensus sequence as a suitable means for the improvement of thestability of isolated variable immunoglobulin domains (Ohage 1999; Ohage1999 and U.S. Pat. No. 5,854,027, hereby incorporated by reference).

Plückthun (EP 0859841) discloses methods for the further optimization ofbinding affinities based on these consensus sequences. The Plückthunpatent also acknowledges the ongoing increase in knowledge concerningantibodies and accordingly aims at including such future findings in thelibrary design. However, no possible further improvements of thesynthetic consensus frameworks are suggested.

The teachings of Winter, Plückthun and others (e.g. Soderlind, WO0175091) have thus tried to create large antibody libraries with a focuson high diversity in the CDRs for selection and application of theselected scFvs under oxidizing conditions. All of these libraries are,however, not optimized for intracellular applications and thus notuseful for selection and applications in a reducing environment, orother conditions which set special requirements on stability andsolubility of the expressed antibody fragment.

The qualities required for antibody fragments to perform well in areducing environment, e.g. the cytoplasm of prokaryotic and eukaryoticcells, are not clear. The application of intracellular antibodies or“intrabodies” is therefore currently limited by their unpredictablebehavior under reducing conditions, which can affect their stability andsolubility properties (Biocca, 1995; Wörn, 2000). Present patentapplications (EP1040201, EP1166121 and WO0200729) and publications(Visintin, 1999) concerning intracellular screening for intrabodiesfocus on the screening technology but do not disclose specific antibodysequences which are functional in eukaryotic cells, in particular inyeast, and, thus, useful for library construction in this context.

Visintin and Tse have independently described the isolation of aso-called intracellular consensus sequence (ICS) (Visintin, 2002; Tse,2002). This sequence was derived from a number of sequences that hadbeen isolated from an antigen-antibody-interaction screen in yeast. Theinput into the intracellular screen was, however, heavily biased due toprior phage-display selection. Thus, all but one of the input-sequencesbelonged to the VH 3 subgroup in the case of Visintin et al. Thepublished consensus sequence ICS is fully identical to the consensussequence for the human VH 3 subgroup described by Knappik (2000) andEP0859841. VH1a consensus sequence

(Seq. Id. No. 14) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTAANYAQKFQGRVTITADESTSTAYMELSSLRSEADTAVYYCARWGGDGFYAMDYWGQGTLVTVSSand VH1b consensus sequence

(Seq Id. No. 15) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWINPNSGGTBNYAQKFQGRVTMTRDTSISTAYMELSSLRSEBDTAVYYCARWGGDGFYAMDYWGQGTLVTVSS60 of the 62 amino acids of the ICS are also identical to the generalhuman VH-domain consensus sequence which was proposed by Steipe as abasis for the construction of variable domains with enhanced stability(U.S. Pat. No. 6,262,238, hereby incorporated by reference). These workswere, in turn, based on earlier sequence collections (i.e., Kabat, 1991and definitions of variable domain subgroups and structural determinants(Tomlinson, 1992; Williams, 1996; Chothia, 1989 and Chothia, 1987).However, because the input to the intrabody selection was so heavilybiased (i.e., in the case of Visintin et al. all but one of the VHdomains was VH3), the isolation of VH3 sequences from intracellularscreening is not particularly surprising. Due to the heavy bias of theirinput library, the work of Tse et al. and Visintin et al. does notprovide a thorough evaluation of the human variable domain repertoire aswould be provided by an unbiased inquiry and as is required to identifythe useful intrabody frameworks present in the human repertoire.

We have previously described a system, which allows for the selection ofstable and soluble intrabodies in yeast, independent of theirantigen-binding specificity (Auf der Maur (2001), WO0148017). Thisapproach allows efficient screening of scFv libraries and the isolationof specific frameworks, which are stable and soluble in the reducingenvironment of the yeast cell. The objective remains to actually isolateframework sequences and use the patterns in a first step to predict whatsequence types would be most stable in the reducing environment and in asecond step identify by analysis, recombination and further in vivo andin vitro experiments the optimal sequence.

BRIEF SUMMARY OF THE INVENTION

The present invention fills a missing link in the field of antibodygeneration. It provides antibody variable domain framework sequenceswith superior characteristics regarding stability and solubility. Theseare crucial features for many relevant applications, such as indiagnostics, therapy or research. These frameworks can be used forgrafting of existing binding-specificities or for the generation ofantibody libraries with high stability and solubility.

ScFv libraries were used for the isolation of frameworks which arestable and soluble in the reducing environment of the yeast cell. Theperformance of the isolated frameworks has subsequently beencharacterized in human cell lines and in in vitro experiments. Thedescribed frameworks can directly serve as acceptor backbones forexisting binding specificities or to construct CDR libraries byrandomization of one or more of the hypervariable loops for use inreducing or otherwise challenging environments. The isolated variabledomain sequences have further been analyzed by alignment to identifypreferred sequence families. From those preferred variable domainsequence families, optimal sequences were chosen based on a structuralanalysis which excludes sequences containing framework residues whichdisturb the immunoglobulin fold. The identified variable domain sequencecandidates were subsequently recombined in all possible variations andthe optimal combinations of variable domains of the light and heavychain were selected by analysis of their performance in yeast, mammaliancells and in vitro.

These optimized scFvs and their constituting variable domain frameworks,as well as other antibody fragments or whole antibodies derived thereof,are ideal as, for example, acceptor backbones for existing bindingspecificities or for the construction of CDR libraries by randomizationof one or more of the hypervariable loops for use in reducing orotherwise challenging environments. Antibodies suitable forintracellular applications are by definition more stable and soluble.Accordingly, their use will also be advantageous in applications outsidethe intracellular environment.

The invention provides compositions comprising frameworks of antibodyvariable domains and single-chain Fv antibody (ScFv) fragments which canbe incorporated into various antibody fragments or whole antibodies.Classes of antibody variable domains fragments are provided which arethe most stable and soluble and thus best suited for intracellularapplications. Specific framework sequences of antibody variable domainsand scFv antibody fragments which show the highest performance inintracellular assays are also provided. The invention also providesspecific framework sequences of antibody variable domains and syntheticcombinations of variable domains of the light and heavy chain in scFvfragments which are, for example, optimal for intracellular applicationsand show an optimal performance in vitro regarding stability andsolubility.

The invention provides single-chain framework reagents that have thegeneral structures:

-   -   NH₂-VL-linker-VH-COOH or    -   NH₂-VH-linker-VL-COOH.

In another embodiment of the invention the single-chain framework may befused to a second protein moiety to yield a fusion construct of thegeneral structure:

-   -   NH₂-VL-linker-VH-second protein-COOH    -   NH₂-second protein-VL-linker-VH-COOH.

The orientation of the VH and VL regions in these fusion constructs maybe reversed.

In another embodiment of the invention the variable domains may beincorporated into a Fab fragment, which may additionally be fused to asecond protein moiety to yield fusion constructs of the generalstructure:

-   -   NH₂-VH-CH-second protein-COOH and NH₂-VL-CL-COOH

The second protein may be fused to either N- or C-terminus of either theheavy or the light chain.

In a preferred embodiment, the second protein of the single-chain or Fabframework fusion construct is a protein which provides a read-out forintracellular assays, either directly or via transcriptional activation.

Another object of the invention is to provide framework classes ofantibody variable domains and sequences of variable domains and scFvswhich are suitable for grafting the hypervariable loops from existingantibodies, for example, in order to obtain antibodies which arefunctional in a reducing or otherwise challenging environment.

Another object of the invention is to provide framework classes ofantibody variable domains and sequences of variable domains and scFvswhich, for example, through randomization of one or more of thehypervariable loops of such frameworks, are suitable for the creation oflibraries for use in a reducing or otherwise challenging environment.

Another object of the invention is the use of the disclosed sequences inthe identification of conserved residues and consensus sequences.

The antibodies or antibody fragments resulting from the use of thedisclosed frameworks can be used as reagents in target validation and intherapy, prevention and diagnosis of human, animal and plant diseases.The antibodies can be used in the form of protein or DNA encoding such aprotein and are not limited to intracellular applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the result of a typical “quality control” screen in yeastassayed by activation of lacZ expression (see, for example, Example 1).The selected, positive clones (black) were identified in severaldifferent screens and the corresponding sequences of the positive clonescan be found in FIGS. 12 and 13. The selected sequences are compared tothe positive control, the very stable lambda-graft (dark grey).

FIG. 2 shows the performance of the frameworks isolated from a typical“quality control” screen in yeast (black) in the human cell line Hela,assayed by the activation of luciferase expression in comparison to thevery stable lambda-graft (dark grey). The positive control Gal4-VP16(white) gives the maximally possible level of transcriptional activationin the system. Luciferase activity has been corrected for transfectionefficiency.

FIG. 3 shows the in vivo performance of the superior frameworkcombinations assayed in yeast by the activation of lacZ expression. Theframework sequences (black) are compared to the positive control (thevery stable lambda-graft (dark grey)). The numbering of the frameworksis as described in FIG. 16.

FIG. 4 shows the in vivo performance of the superior frameworkcombinations assayed in the human cell line Hela by the activation ofluciferase expression and illustrated in comparison to the very stablelambda-graft (dark grey). The positive control, Gal4-VP16 (white) givesthe maximal possible level of transcriptional activation in the system.Luciferase activity has been corrected for transfection efficiency.

FIG. 5 shows the in vivo performance of the superior frameworkcombinations assayed by the amount of soluble protein produced in thecytoplasm of yeast strain S. cerevisiae JPY9.

FIG. 6 shows the expression behavior of selected framework combinationsin the periplasm of E. coli. The arrow indicates the location of theband corresponding to the scFv frameworks.

FIG. 7 shows the in vivo performance of selected superior frameworkcombinations assayed in three human cell lines (Hela, (black), Saos-2(dark grey) and HEK 293 (white)), by the activation of luciferaseexpression and illustrated in comparison to the very stablelambda-graft. The positive control Gal4-VP16 gives the maximal possiblelevel of transcriptional activation in the system. Luciferase activityhas been corrected for transfection efficiency.

FIG. 8 represents the resistance towards aggregation at 37° C. ofselected framework combinations as quantified by the amount of monomericprotein present before and after incubation as indicated in PBS-buffer.Panel A is representative for frameworks 2.4 and 5.2 and panel B forframeworks 4.4, 6.4 and 7.3.

FIG. 9 represents the resistance towards protease degradationaggregation in human serum at 37° C. of selected framework combinations,quantified by the amount of soluble full-length protein present beforeand after prolonged incubation.

FIG. 10 shows the in vivo performance of two selected binders on thenovel framework 7.3 in the Fab-context, assayed in yeast interactionassay by the activation of lacZ expression. Expression of the Fab-chainsis from a bi-directional galactose-inducible promoter, on either anars/cen or a 2 micron vectors. Expression from the Fab vector yields theantibody light chain and a VH-CH1-Gal4-AD fusion protein. Binders aredirected against human Polo-like kinase1 (hPLK1). Binding to the targetis compared with the unspecific binding to an unrelated antigen and thebinding of the un-randomized framework 7.3. Note that the correspondingscFv that have been included for reference are expressed from an actinpromoter (2 micron).

FIG. 11 shows the in vivo performance of the scFv frameworks in theFab-context assayed by the amount of soluble protein produced in thecytoplasm of the yeast strain JPY9. Expression of the Gal4-AD-scFvfusion (actin/2 micron) is compared with the expression of thecorresponding Fab-construct, and with the parent framework 7.3 as a Fab,both from two different vectors (Gal-inducible, ars/cen and 2 micron).Expression from the Fab vector yields the antibody light chain and aVH-CH1-Gal4-AD fusion protein, which is detected in this blot.

FIG. 12 shows an alignment of all VH-domain framework sequences selectedfrom various “quality control” screens in yeast.

FIG. 13 shows an alignment of all VL-domain framework sequences selectedfrom various “quality control” screens in yeast.

FIG. 14 shows an alignment of randomly picked sequences from thelibrary.

FIG. 15 shows a statistical analysis of the sub-class frequency for VH-and VL-domains in the sequences isolated with the “quality control”system. Only those sequences were considered which were subsequentlyfound to be positive in the quantitative yeast assay. The selectedsequences are compared with the unselected library as determined from alimited number of random sequences (FIG. 14).

FIG. 16 shows the sequences used for further recombination andevaluation of the best combinations in scFvs and their respectiveabbreviations (abb.), sources and sub-family.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

As used herein, “identity” refers to the sequence similarity between twopolypeptides, molecules or between two nucleic acids. When a position inboth of the two compared sequences is occupied by the same base or aminoacid monomer subunit (for instance, if a position in each of the two DNAmolecules is occupied by adenine, or a position in each of twopolypeptides is occupied by a lysine), then the respective molecules arehomologous at that position. The “percentage identity” between twosequences is a function of the number of matching positions shared bythe two sequences divided by the number of positions compared×100. Forinstance, if 6 of 10 of the positions in two sequences are matched, thenthe two sequences have 60% identity. By way of example, the DNAsequences CTGACT and CAGGTT share 50% homology (3 of the 6 totalpositions are matched). Generally, a comparison is made when twosequences are aligned to give maximum homology. Such alignment can beprovided using, for instance, the method of Needleman et al., J. Mol.Biol. 48: 443-453 (1970), implemented conveniently by computer programssuch as the Align program (DNAstar, Inc.).

“Similar” sequences are those which, when aligned, share identical andsimilar amino acid residues, where similar residues are conservativesubstitutions for, or “allowed point mutations” of, corresponding aminoacid residues in an aligned reference sequence. In this regard, a“conservative substitution” of a residue in a reference sequence is asubstitution by a residue that is physically or functionally similar tothe corresponding reference residue, e.g., that has a similar size,shape, electric charge, chemical properties, including the ability toform covalent or hydrogen bonds, or the like. Thus, a “conservativesubstitution modified” sequence is one that differs from a referencesequence or a wild-type sequence in that one or more conservativesubstitutions or allowed point mutations are present. The “percentagepositive” between two sequences is a function of the number of positionsthat contain matching residues or conservative substitutions shared bythe two sequences divided by the number of positions compared×100. Forinstance, if 6 of 10 of the positions in two sequences are matched and 2of 10 positions contain conservative substitutions, then the twosequences have 80% positive homology.

“VH domain” refers to the variable part of the heavy chain of animmunoglobulin molecule.

“VL domain” refers to the variable part of the light chain of animmunoglobulin molecule.

VH or VL “subtype” refers to the subtype defined by the respectiveconsensus sequence as defined in Knappik (2000). The term “subfamily” or“subclass” is used as synonym for “subtype”. The term “subtype” as usedherein refers to sequences sharing a high degree of identity andsimilarity with the respective consensus sequence representing theirsubtype. Whether a certain variable domain sequence belongs to a“subtype” is determined by alignment of the sequence with either allknown human germline segments of the respective domain, or the definedconsensus sequences and subsequent identification of the greatesthomology. Methods for determining homologies and grouping of sequencesby using search matrices, such as BLOSUM (Henikoff 1992) are well knownto the person skilled in the art.

“Amino acid consensus sequence” as used herein refers to an amino acidsequence, which can be generated using a matrix of at least two orpreferably more aligned amino acid sequences, and allowing for gaps inthe alignment, it is possible to determine the most frequent amino acidresidue at each position. The consensus sequence is that sequence whichcomprises the amino acids which are most frequently represented at eachposition. In the event that two or more amino acids are equallyrepresented at a single position, the consensus sequence includes bothor all of those amino acids.

The amino acid sequence of a protein can be analyzed at various levels.For example, conservation or variability could be exhibited at thesingle residue level, multiple residue level, multiple residue with gapsetc. Residues could exhibit conservation of the identical residue orcould be conserved at the class level. Examples of amino acid classesinclude polar but uncharged R groups (Serine, Threonine, Asparagine andGlutamine); positively charged R groups (Lysine, Arginine, andHistidine); negatively charged R groups (Glutamic acid and Asparticacid); hydrophobic R groups (Alanine, Isoleucine, Leucine, Methionine,Phenylalanine, Tryptophan, Valine and Tyrosine); and special amino acids(Cysteine, Glycine and Proline). Other classes are known to one of skillin the art and may be defined using structural determinations or otherdata to assess substitutability. In that sense a substitutable aminoacid could refer to any amino acid which could be substituted andmaintain functional conservation at that position.

“Polynucleotide consensus sequence” as used herein refers to anucleotide sequence, which can be generated using a matrix of at leasttwo or preferably more aligned nucleic acid sequences, and allowing forgaps in the alignment, it is possible to determine the most frequentnucleotide at each position. The consensus sequence is that sequencewhich comprises the nucleotides which are most frequently represented ateach position. In the event that two or more nucleotides are equallyrepresented at a single position, the consensus sequence includes bothor all of those nucleotides.

“Structural sub-element” as used herein refers to stretches of aminoacid residues within a protein or polypeptide that correspond to adefined structural or functional part of the molecule. These can beloops (i.e. CDR loops of an antibody) or any other secondary orfunctional structure within the protein or polypeptide (i.e., domains,α-helices, β-sheets, framework regions of antibodies, etc.). Astructural sub-element can be identified using known structures ofsimilar or homologous polypeptides, or by using the above mentionedmatrices of aligned amino acid sequences. Here the variability at eachposition is the basis for determining stretches of amino acid residueswhich belong to a structural sub-element (e.g. hypervariable regions ofan antibody).

“Sub-sequence” as used herein refers to a genetic module which encodesat least one structural sub-element. It is not necessarily identical toa structural sub-element.

“Antibody CDR” as used herein refers to the complementarity determiningregions of the antibody which consist of the antigen binding loops asdefined by Kabat et al. (1991). Each of the two variable domains of anantibody Fv fragment contain, for example, three CDRs.

“Antibody” as used herein is a synonym for “immunoglobulin”. Antibodiesaccording to the present invention may be whole immunoglobulins orfragments thereof, comprising at least one variable domain of animmunoglobulin, such as single variable domains, Fv (Skerra, 1988), scFv(Bird, 1988; Huston, 1988), Fab, (Fab′)2 or other fragments well knownto a person skilled in the art.

“Antibody framework” as used herein refers to the part of the variabledomain, either VL or VH, which serves as a scaffold for the antigenbinding loops of this variable domain (Kabat et al., 1991).

Rationally engineered scFv fragments have demonstrated a clearcorrelation between the thermodynamic stability of a scFv fragment andits in vivo performance (Wörn, 2000; Auf der Maur, 2001). Using arecently developed system named “Quality Control” (Auf der Maur, 2001),specific antibody variable domain framework sequences which are suitablefor intracellular applications have been isolated (FIG. 12 and FIG. 13),characterized (FIGS. 1 and 2) and further improved (FIG. 3 to 9 and FIG.14). As observed in our previous experiments, well performing frameworksselected in the intracellular assay show a high in vitro stability asdemonstrated by their resistance to aggregation and protease degradationat 37° C. (FIGS. 8 and 9). Moreover, a pattern emerged which allows aselection of frameworks for intracellular applications on a more generalbasis, depending on their framework subfamily (FIG. 15). Specificantibody variable domain sequences useful for intracellular applicationsare disclosed here, as well as the general pattern. This allows, on theone hand, the use of these sequences as framework donors in graftingexperiments to obtain functional intrabodies which retain the bindingspecificity of the loop donor. Additionally, antibody libraries can beconstructed using the disclosed sequences as frameworks. Such librariesare suitable for intracellular selection systems under reducingconditions, such as those in prokaryotic and eukaryotic cells.Additionally, the disclosed sequences may be used to identify, forexample, conserved sequences or residues or motifs. The grafting ofstructural sub-elements, for example, those of the binding loops of anantibody (e.g. Jung, 1997), as well as the making of libraries ofantibodies or fragments thereof (e.g. Vaughan, 1996; Knappik, 2000) hasbeen described in detail and is well known to a person skilled in theart.

Because intracellular applications expose the antibody fragments to veryunfavorable conditions (i.e. increased temperatures, reducingenvironment), the sequences disclosed in the present invention haveacquired features that make them resistant to the most adverseconditions. Therefore, when compared to “average” sequences, thedisclosed sequences are of outstanding stability and solubility as isdemonstrated by their resistance towards aggregation and proteasedegradation (FIGS. 8 and 9). These features, together with theirexcellent expression yield make the disclosed antibody frameworksequences uniquely suitable not only for intracellular use, butespecially for all therapeutic and diagnostic applications where longhalf-life, robustness, and ease of production are of great concern.

The present invention enables the design of polypeptide sequencescomprising at least the variable part of an antibody that are useful forapplications in a reducing or otherwise challenging environment. In afirst embodiment, the invention provides a collection of antibodyframework sequences useful for intracellular applications (FIG. 12 andFIG. 13). In a first step, a library of diverse sequences is screenedindependent of binding affinity using the Quality control system inyeast. The isolated sequences can be evaluated for their intracellularperformance in yeast and in mammalian cells (FIGS. 1 and 2).

In one embodiment of the invention, the collection of isolated sequencesis analyzed by alignment to identify the antibody variable domainsub-classes and consensus sequences that are suitable for intracellularapplications.

In a further preferred embodiment of the invention, the collection ofantibody framework sequences described above is further analyzed byalignment to each other and grouping into sub-families. All frameworksbelonging to one sub-type are compared regarding their intracellularperformance in yeast and in mammalian cells (FIGS. 1 and 2, as anexample) and regarding the occurrence of negative, neutral or positiveexchanges in their amino-acid sequence relative to the respectivesub-type consensus. A person skilled in the art can distinguish betweenpositive, neutral and negative changes based on the structuralenvironment of the particular exchanged residue in the immunoglobulindomain. Subsequently, framework sequences of variable antibody domainsare chosen which show the best intracellular performance and which aredevoid of negative exchanges compared to their respective sub-typeconsensus. Preferably, sequences are selected which further containamino-acid exchanges which are considered positive.

In a further preferred embodiment, the selected antibody variabledomains of the heavy and the light chain are subsequently recombined inall possible combinations into scFv fragments, in order to identify thecombinations with the highest stability and solubility. To this end thenovel, recombined scFv fragments are evaluated for their performanceunder reducing conditions in intracellular interaction assays in yeast(FIG. 3) and in mammalian cell lines (FIGS. 4 and 7) and for solubleintracellular expression in yeast (FIG. 5). Promising combinations arefurther evaluated for their behavior under oxidizing conditions byanalyzing the periplasmic expression yield in E. coli (FIG. 6), theresistance to aggregation at elevated temperatures (FIG. 8) and theresistance to aggregation and protease degradation upon prolongedincubation in human serum at 37° C. (FIG. 9). These data are used toidentify the scFv framework best suitable for any specific application,either intracellular, or under oxidizing conditions.

The selected and optimized framework sequences disclosed herein have asignificant advantage not only in intracellular applications, but in allapplications which can profit from increased stability and/or solubilityof the scFv. Examples are the long-term storage at high concentrationsrequired for diagnostic applications, and prolonged functional half-lifein serum at 37° C. (as required, for example, in therapeuticapplications).

According to one aspect of the present invention, there is provided anintrabody framework comprising a single-chain framework having thegeneral structure:

-   -   NH₂-VL-linker-VH-COOH; or    -   NH₂-VH-linker-VL-COOH    -   wherein the VH framework is of subtype 1a, 1b or 3.

In another embodiment, the orientation of the VH and VL regions isreversed in the single chain framework described above.

According to one aspect of the present invention, there is provided anintrabody framework comprising a single-chain framework having thegeneral structure:

-   -   NH₂-VL-linker-VH-COOH; or    -   NH₂-VH-linker-VL-COOH    -   wherein the VH framework is of subtype 1a, 1b or 3 and the VL        framework is of subtype λ1, λ3 or κ1.

In another embodiment, the invention provides a single-chain frameworkfused to a second protein moiety to yield a fusion construct of thegeneral structure:

-   -   NH₂-VL-linker-VH-second protein-COOH; or    -   NH₂-second protein-VL-linker-VH-COOH    -   wherein the VH framework is of subtype 1a, 1b or 3 and the VL        framework is of subtype λ1, λ3 or κ1.

In another embodiment, the orientation of the VH and VL regions in thesefusion constructs may be reversed.

In another embodiment, the variable domains may be incorporated into aFab fragment which may additionally be fused to a second protein moietyto yield fusion constructs of the general structure:

-   -   NH₂-VH-CH-second protein-COOH and NH₂-VL-CL-COOH

The second protein may be fused to either N- or C-terminus of either theheavy or the light chain.

As disclosed herein, there is a very strong preference in intracellularapplications for VH framework of the subtype 3, but also for 1a and 1b.Regarding the light chain variable domain (VL), there is a clearpreference by numbers for frameworks of the kappa 1 type, but lambda 1and 3 are also enriched. These framework subtypes, i.e. VH 1a, 1b and 3combined with a kappa 1, lambda 1 or 3 VL domain are therefore bestsuited for intracellular use and other applications which require thefolding properties of the scFv. Therefore, in order to reduce the amountof molecules which are not functional in the reducing environment,libraries for intracellular screening systems should preferentially beconstructed from a mixture of these framework subtypes.

In a preferred embodiment, the VH domain of the antibody fragments ofthe invention is of the subgroup 1a, 1b or 3.

In a preferred embodiment, the VL domain of the antibody fragments ofthe invention is of the subgroup kappa1, lambda 1 or 3.

In a preferred embodiment, antibody fragments used as frameworks areselected from the group consisting of: 1.1, 2.1, 3.1, 4.1, 5.1, 1.2,2.2, 3.2, 4.2, 5.2, 1.3, 2.3, 3.3, 4.3, 5.3, 7.3, 1.4, 2.4, 3.4, 4.4,5.4, and 6.4 as described in FIG. 16.

In one embodiment of the invention, at least two and preferably moreframeworks are identified and then analyzed. A database of the proteinsequences may be established where the protein sequences are alignedwith each other. The alignment can then be used to define, for example,residues, sub-elements, sub-sequence or subgroups of framework sequenceswhich show a high degree of similarity in both the sequence and, if thatinformation is available, in the structural arrangement.

The length of the sub-elements is preferably, but not exclusivelyranging between 1 amino acid (such as one residue in the active site ofan enzyme or a structure-determining residue) and 150 amino acids (forexample, whole protein domains). Most preferably, the length rangesbetween 3 and 25 amino acids, such as most commonly found in CDR loopsof antibodies.

In another embodiment, consensus nucleic acid sequences, which arepredicted from the analysis are synthesized. This can be achieved by anyone of several methods well known to the practitioner skilled in theart, for example, by total gene synthesis or by PCR-based approaches.

In another embodiment, the nucleic acid sequences are cloned into avector. The vector could be a sequencing vector, an expression vector ora display (e.g. phage display) vector, all which are well known to thoseof skill in the art. A vector could comprise one nucleic acid sequence,or two or more nucleic sequences, either in different or the sameoperon. In the last case, they could either be cloned separately or ascontiguous sequences.

In one embodiment, the polypeptides have an amino acid patterncharacteristic of a particular species. This can for example be achievedby deducing the consensus sequences from a collection of homologousproteins of just one species, most preferably from a collection of humanproteins.

A further embodiment of the present invention relates to fusion proteinsby providing for a DNA sequence which encodes both the polypeptide, asdescribed above, as well as an additional moiety.

In further embodiments, the invention provides for nucleic acidsequences, vectors containing the nucleic acid sequences, host cellscontaining the vectors, and polypeptides obtainable according to themethods described herein.

In a further embodiment, the invention provides for synthesizing orotherwise placing restriction sites at the end of the nucleic acidsequences of the invention allowing them to be cloned into suitablevectors.

In a further preferred embodiment, the invention provides for vectorsystems being compatible with the nucleic acid sequences encoding thepolypeptides. The vectors comprise restriction sites, which would be,for example, unique within the vector system and essentially unique withrespect to the restriction sites incorporated into the nucleic acidsequences encoding the polypeptides, except for example the restrictionsites necessary for cloning the nucleic acid sequences into the vector.

In another embodiment, the invention provides for a kit, comprising oneor more of the list of nucleic acid sequences, recombinant vectors,polypeptides, and vectors according to the methods described above, and,for example, suitable host cells for producing the polypeptides.

All of the above embodiments of the present invention can be effectedusing standard techniques of molecular biology known to one skilled inthe art.

In another embodiment, the nucleic acid sequence is any sequence capableof encoding the polypeptides of the invention.

In another embodiment, the inventive nucleic acids are used in genetherapy.

In another embodiment, the single chain framework is a variant of anyone of sequences. 1.1, 2.1, 3.1, 4.1, 5.1, 1.2, 2.2, 3.2, 4.2, 5.2, 1.3,2.3, 3.3, 4.3, 5.3, 7.3, 1.4, 2.4, 3.4, 4.4, 5.4, 6.4 (FIG. 16), where“variant” as used herein refers to a sequence that exhibits 90% orgreater identity, while maintaining enhanced stability.

In another embodiment, the single chain framework is a derivative of anyone of sequences 1.1, 2.1, 3.1, 4.1, 5.1, 1.2, 2.2, 3.2, 4.2, 5.2, 1.3,2.3, 3.3, 4.3, 5.3, 7.3, 1.4, 2.4, 3.4, 4.4, 5.4, 6.4 (FIG. 16) where“derivative” as used herein refers to a sequence that maintains onlythose amino acids that are critical to the function and stability of themolecule. Isolated neutral or positive exchanges in the framework asdescribed in example 3, are not considered to be relevant change to theantibody frameworks of the present invention.

In a preferred embodiment of the invention, the single chain frameworkis fused to a second protein, wherein that protein provides a read-outfor intracellular assays. The read-out can be either direct, for examplein the form of a fusion to a detectable protein, e.g. GFP (greenfluorescent protein), enhanced blue fluorescent protein, enhanced yellowfluorescent, protein enhanced cyan fluorescent protein which can beobserved by fluorescence, or other fusion partners with differentdetection methods. Alternatively, a read-out can be achieved throughtranscriptional activation of a reporter gene, where the fusion partnerin the scFv-fusion protein is either a transcriptional activator, suchas the Gal4 activation domain, or a DNA-binding protein, such as theLexA- or Gal4 DNA-binding domain, which activates the transcription of areporter gene of an enzyme, such as β-galctosidase, luciferase,α-galactosidase, β-glucuronidase, chloramphenicol acetyl transferase andothers, which in turn provide a read-out. Fusion proteins, which providea read out are well known to one of skill in the art.

Another embodiment of the invention is an antibody comprising aframework described herein.

Another embodiment of the invention is the use of the antibody of theinstant invention.

A further preferred embodiment of the invention is the use of thedescribed framework classes of antibody variable domains and sequencesof variable domains and scFvs for grafting of hypervariable loops fromexisting antibodies, in order to obtain antibodies which are functionalin a reducing or otherwise challenging environment.

Another further preferred embodiment of the invention is the use of thedescribed framework classes of antibody variable domains and sequencesof variable domains and scFvs, for example through randomization of oneor more of the hypervariable loops of such frameworks, for the creationof libraries for applications in a reducing or otherwise challengingenvironment.

As would be apparent to one of ordinary skill in the art, the inventivemolecules described herein may be used in diagnostic and therapeuticapplications, target validation and gene therapy.

The invention may be illustrated by the following examples, which arenot intended to limit the scope of the invention in any way.

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The invention is further illustrated in the following non-limitingexamples.

EXAMPLE 1 Selection of Intrabody Frameworks Through Screening of a HumanLibrary in the “Quality Control” System in Yeast

Screening with the “quality control” system for stable frameworks wasessentially performed as described in detail by Auf der Maur (WO0148017,Auf der Maur 2001, each hereby incorporated by reference).

The plasmids for expression of the scFv-fusion constructs for screeningin yeast were derived from pESBA-Act (Wörn, 2000). It contains the yeastTRP1 gene for transformation selection in S. cerevisiae and the 2 micronorigin of replication to ensure high copy numbers. Moreover it has aconstitutive actin promoter for strong expression and the GAL11transcriptional termination sequence, separated by a multiple cloningsite. For handling in bacterial systems, it also has a bacterial originof replication and the amp resistance gene.

The Gal4 activation domain (AD amino acids 768-881) was originallyamplified by PCR using pGAD424 (Clontech) as template with primersincluding the SV40 T-antigen nuclear localization signal N-terminal tothe Gal4-AD. The DNA-fragments encoding amino acids 263-352 of Gal11Pwere amplified by PCR and cloned in frame, N-terminal to theSV40-NLS-Gal4-AD-construct. The human scFv library, amplified from humanspleen-cell cDNA as described elsewhere (Welschhof, 1995; Krebber, 1997;de Haard, 1999), was cloned in frame, N-terminal to this fusionconstruct via SfiI-sites, and in the orientation V_(L)-linker-V_(H)where the linker has the sequence (GGGS)₄. Expression thus yields afusion protein of the general structure scFv-Gal11p-SV40 NLS-Gal4AD.

Screening was carried out in the yeast strain S. cerevisiae YDE172 (MATαura3-52 leu2Δ1 trp1d63 his3Δ200 lys2Δ 385 gal4Δ 11) (Auf der Maur,2001), which was derived from the strain JPY9 (Escher, 2000) byintegrating the divergently oriented LacZ and HIS3 reporter genes underthe control of the natural UAS_(G) from Gal1-GAL10 regulatory sequencesinto the his3Δ200 locus. Transcriptional activation of the reportersystem is mediated by the Gal4-AD moiety of the scFv-fusion construct,following the specific interaction of its Gal11P moiety with theGal4-DNA-binding-domain (DBD, amino acids 1-100). The Gal4-DBD isprovided by expression from a second plasmid, pMP83. It contains theyeast LEU2 gene for transformation selection in S. cerevisiae and theARS CEN origin of replication. Moreover, it has a constitutive actinpromoter for strong expression and the GAL11 transcriptional terminationsequence. For handling in bacterial systems, it also has a bacterialorigin of replication and the amp resistance gene.

For screening, the yeast strain S. cerevisiae YDE172 was co-transformedwith a scFv-library as fusion construct on the pESBA-Act2 vector whilethe pMP83-vector provided the Gal4-DBD. A standard lithium acetatetransformation protocol was used (Agatep, 1998). Followingtransformation, the cells were plated on drop-out plates(-Trp/-Leu/-His) containing 80 mM 3-aminotriazole. Colonies were pickedafter 3 days incubation at 30° C. and restreaked on drop-out plates(-Trp/-Leu/-His) containing 80 mM 3-aminotriazole. Those that re-grewwere tested for LacZ-expression by development of blue color in a filterassay on plates containing the substrate X-Gal. Positive clones weretaken for further analysis involving isolation of the scFv-carryingplasmid from yeast, transformation into E. coli DH5α, isolation ofplasmid from single colonies of E. coli and re-transformation intofreshly prepared yeast strain S. cerevisiae YDE172 for the assay asdescribed below. All methods were performed according to standardprocedures, well known to a person of ordinary skill in the art.

In addition, a modified screening procedure was used were the scFv wasdirectly fused to both a DNA-binding domain (LexA amino acids 1-202) andan activation domain (Gal4, amino acids 768-881) to yield a fusionconstruct of the following structure: scFv-LexA-NLS-Gal4AD. The plasmidsfor expression of the scFv-fusion constructs for screening in yeast werederived from pESBA-Act2. It contains the yeast TRP1 gene fortransformation selection in S. cerevisiae and the 2 micron origin ofreplication to ensure high copy numbers. Moreover, it has a constitutiveactin promoter (for strong expression) and the GAL11 transcriptionaltermination sequence separated by a multiple cloning site. For handlingin bacterial systems, it also has a bacterial origin of replication andthe amp resistance gene.

Screening was carried out in the yeast strain S. cerevisiae ImmunaLHB(MATα ura3-52 leu2Δ1 trp1d63 his3Δ200 lys2Δ 385) which was derived fromthe strain JPY5 by integrating the divergently oriented LacZ and HIS3reporter genes under the control of a bi-directional promoter with sixLexA-binding sites (integrating reporter plasmid pDE200, Escher 2000)into the his3Δ200 locus and by integrating the LEU2 reporter gene underthe control of a promoter with eight LexA-binding sites (derived fromEGY48) into the leu2Δ1 locus. Transcriptional activation of the reportersystem is mediated by the Gal4-AD moiety of the scFv-fusion construct.Screening was carried out essentially as described above using drop-outmedium (-Trp/-Leu/-His) and 3-aminotriazole concentrations up to 40 mM.

EXAMPLE 2 Evaluation of in Vivo Performance

a) In Yeast

For quantitative analysis of the performance of the selected frameworksin yeast (FIGS. 1 and 3), S. cerevisiae-strain Immuna LHB wastransformed with the isolated scFvs as LexA-Gal4-AD-fusion constructs onthe pESBA-Act2 vector by following a standard lithium acetatetransformation protocol (Agatep, 1998). Following transformation, thecells were plated on drop-out plates (Trp). 2 ml overnight-cultures indrop-out medium (-Trp) were inoculated in duplicates from streakscontaining several colonies and grown at 30° C. Cultures were diluted in1 ml drop-out medium (-Trp) to an optical density at 600 nm (OD600) of0.7. They were then grown at 30° C. for 2 h. For the assay, 100 μl cellculture were taken, mixed with 900 μl buffer, 45 μl Chloroform and 30 μl0.1% SDS, vortexed and incubated at room temperature for 5 minutes. Thecolor development was initiated by the addition of 0.2 ml ONPG (4 mg/ml)and stopped with 0.5 ml Na₂CO₃ (1 M). The activity was calculated bytaking into account the OD600 of the assay culture, as well as theincubation time of the color development and the culture volume used

Clones that were at least equal to or better than the positive control(the very stable lambda-graft described before (Worn, 2000; Auf derMaur, 2001)) were sequenced to identify the framework subtype (frameworksubtype definitions according to Tomlinson, (1992), Cox, (1994) andWilliams, (1996)). Sequencing revealed a striking preference for certainframework subtypes. For the heavy chain variable domain (VH), frameworksubtypes 2 and 6 were never found and 4 was markedly reduced among thepositive clones. Corrected for the performance of the isolated sequencesin the yeast intracellular assay, there is a very strong preference forVH framework of the subtype 3, but also for 1a and 1b in intracellularapplications. Regarding the light chain variable domain (VL) there is aclear preference for frameworks of the kappa 1, lambda 1 and lambda 3sub-types (FIG. 15).

These framework subtypes, i.e. VH 1a, 1b and 3 combined with a kappa 1,lambda 1 and lambda 3 VL domain are therefore best suited forintracellular use and other applications with stringent requirementsconcerning the folding properties of the scFv. Libraries forintracellular screening systems should, for example, preferentially beconstructed from a mixture of these framework subtypes only, to reducethe amount molecules which are not functional in the reducingenvironment.

b) In Mammalian Cells

Hela cell line was used for quantitative analysis of the performance ofthe selected frameworks in human cells (FIGS. 2, 4 and 7). Theluciferase reporter gene was provided from a co-transfected pGL3(Promega) reporter plasmid containing the luciferase under the controlof the natural Gal4 UAS. The mammalian expression vectors used fortransient transfection contains the Gal4 (1-147) fused on the C-terminusto the VP16-AD under the control of a CMV promoter. The isolated scFvswere cloned in frame, C-terminal to a Gal4(1-147)-VP16-fusion to yield aGal4(1-147)-VP16-scFv-fusion protein upon expression. Cells werecultured in DMEM supplemented with 2.5% FCS and 2 mM l-glutamine.Transient transfections were carried out according to thePolyfect-protocol (Qiagen) in 60 mm tissue culture plates using 0.01-0.1μg of the vector containing the scFv-construct, 0.5 μg of a CMVpromoter-driven Gal4 (1-147)-VP16-scFv expression plasmid and 0.5 μg ofa LacZ expression vector as reference for transfection efficiency. Cellswere harvested 24-48 hours after transfection, resupended in 1000 μlbuffer and lysed by three freeze-thaw-cycles. The cell lysate wascentrifuged and the supernatant assayed for luciferase activity usingluciferase assay solution (Promega) and for LacZ activity according tothe standard protocol. The obtained luciferase activity was correctedwith the LacZ activity to account for the variation in transfectionefficiency.

EXAMPLE 3 Multiple Alignment and Analysis of the Sequence Comparison

To elucidate the general pattern of framework sequences suitable forintracellular applications, all positive clones (i.e. those that growunder selective conditions in the quality control system) were isolatedand the part coding for the scFvs was sequenced. Subsequently, the scFvsequences were divided in their light and heavy-chain component to allowalignment of the respective domains (FIG. 12 and FIG. 13) according tothe structural adjusted numbering scheme of immunoglobulin domains byHonegger (2001).

To allow evaluation of the obtained data, an alignment representing theunselected library was generated (FIG. 14). In order to obtainunselected sequences, the library was transformed in E. coli cells whichdo not express the scFv-genes and clones were picked at random forplasmid isolation and sequencing of the scFv-sequence. The librarycovers the human antibody repertoire as expected and thus has no biastowards specific subgroups, other than expected by the expressionpattern generally found in humans.

The VH and VL sequences were grouped according to their subgroup.Changes to the subgroup-specific consensus sequence were highlighted. Aperson skilled in the art can distinguish between positive, neutral andnegative changes based on the structural environment of the particularexchanged residue (e.g. Honegger, 2001). An exchange of a residuebelonging to a particular group of amino acids to a residue of the samegroup is in general validated as a neutral exchange. An exchange of aresidue belonging to the group of hydrophobic amino acid pointing intothe hydrophobic core of the protein to one amino acid of the group ofpolar but uncharged or positively or negatively charged amino acidswould be highly unfavorable because unsatisfied hydrogen donor/acceptorsites disturb tight packing of the hydrophobic core. Such a change istherefore considered negative. An exchange of a residue belonging to thegroup of polar but uncharged residues at the surface of theimmunoglobulin domain to an amino acid of the group of positively ornegatively charged residues is highly favorable as the solubility of theprotein is increased. Such a change is therefore validated positively,whereas the exchange from a polar to a hydrophobic residue is highlyunfavorable as the solubility of the protein is decreased and istherefore validated negatively. At positions with a conserved positivephi-angle, an exchange of any amino acid to glycine is validatedpositively whereas an exchange of gylcine to any amino acid is validatednegatively because glycine is the only amino acid which is able to forma positive phi-angle. The loss of a conserved salt bridge betweenpositions 45-53, 45-100, 77-100 and 108-137 because of an exchange froman amino acid of the group of positively or negatively charged residuesto an uncharged amino acid results in a decreased thermodynamicstability and is therefore considered negative.

Finally, we chose 7 VL domains and 4 VH domains that were preferentiallyselected during the quality control (i.e. showing the least negative andmost positive exchanges from the consensus sequence and cover thesubgroups) and that each show high in vivo performance in yeast. Thesequences are summarized in FIG. 16 and include two Vκ1 (k I 27 (1.x)and k III 25(2.x)), two Vκ3 (k IV 103 (3.x) and k IV135 (5.x)), one Vλ1(k IV 107 (4.x)), two Vλ3 (a33 (7.x) and a43 (6.x)), one VH1b (a33(x.3)) and three VH3 (a fw10 (x.2), a43 (x.4) and a44 (x.1)). These VLand VH domains were shuffled giving 22 novel combinations in the scFvformat (1.1, 2.1, 3.1, 4.1, 5.1, 1.2, 2.2, 3.2, 4.2, 5.2, 1.3, 2.3, 3.3,4.3, 5.3, 7.3, 1.4, 2.4, 3.4, 4.4, 5.4, 6.4).

EXAMPLE 4 Evaluation of In Vivo Performance of Shuffled Domains

a) Performance in an Intracellular Assay in Yeast and Mammalian Cells

The 22 combinations were tested for their in vivo performance in yeastand mammalian cells as described in example 2 (FIGS. 3 and 4).

b) Expression of Soluble Protein Under Reducing Conditions in Yeast

To compare the yields of soluble protein upon expression under reducingconditions, the selected frameworks were expressed as a fusion to Gal4AD in the cytoplasm of yeast S. cerevisiae. The fusion constructs on thepESBA-Act2 vector had the general structure Gal4 AD-scFv. They weretransformed as described above into the yeast S. cerevisiae strain JPY9and plated on -Trp, drop-out plates.

5 ml overnight-cultures in drop-out medium (-Trp) were inoculated fromstreaks containing several colonies and grown at 30° C. Cultures werediluted in 50 ml drop-out medium (-Trp) to an optical density at 600 nm(OD600) of 0.5. They were grown at 30° C. for 5 h. For the native cellextract, 2.5 ml cell culture normalized to an OD600 of 3 were harvestedby centrifugation, frozen in liquid nitrogen and subsequentlyresuspended in 75 μl Y-PER (Pierce) containing protease inhibitor(PMSF). The resuspended cell pellet was vortexed shortly and incubated(slightly shaking) at 20° C. for 20 min. Insoluble and aggregatedmaterial was pelleted at maximal speed in an eppendorf centrifuge at 4°C. for 10 min. The supernatant was mixed with loading dye, heated to100° C. for 5 min. and separated on a 12% SDS-PAGE. The soluble Gal4AD-scFv fusion constructs were visualized by western blotting viadetection of the Gal4-moiety with an anti-Gal4AD monoclonal mouseantibody (Santa Cruz Biotechnology) as a primary antibody and ananti-mouse-peroxidase conjugate (Sigma) as secondary antibody and usinga chemoluminescent substrate (Pierce) (FIG. 5). SDS-PAGE and westernblotting procedures are well known to a person of ordinary skill in theart.

c) Expression Behavior in the Periplasm of E. coli

For evaluation of periplasmic expression behavior in E. coli (FIG. 6),isolated scFvs-frameworks were cloned in a bacterial vector harbouringthe cam resistance gene (catR) and the lacI repressor gene (Krebber,1997), with a N-terminal pelB-leader sequence and a C-terminal his-tagunder the control of the lac promoter/operator. Competent E. coli JM83were transformed with these plasmids. 50 ml dYT-medium containing 35mg/l chloramphenicol in shaking flasks was inoculated 1:40 with anover-night culture and incubated at 30° C. Cells were induced at anOD600 of 0.8 with 1 mM IPTG and harvested after 3 hours of induction bycentrifugation. The pellet was resuspended in 50 mM Tris, pH 7.5, 500 mMNaCl and normalized to an OD600 of 10. Samples of each scFv fragmentswere analyzed either directly (total extract) or after sonificationfollowed by centrifugation (soluble fraction) by SDS-PAGE. The amount ofsoluble protein was estimated from the Coomassie-stained gel.

EXAMPLE 5 Detailed Evaluation of 5 Combinations with Superior Propertiesfor Extracellular Use

Five combinations were chosen as examples which show good performanceboth in yeast and mammalian intracellular assays, yield soluble proteinduring expression in yeast and E. coli, and cover the subgroups whichwere preferentially selected during the quality control (2.4, 4.4, 5.2,6.4 and 7.3, see FIG. 16 for details). We analysed these combinations ingreater detail to further evaluate their use under reducing, as well asoxidizing conditions.

a) Performance in an Intracellular Assay in Different Mammalian Cells

The quantitative analysis of the performance of the five combinations inhuman cells was carried out using Hela cells and in addition using thehuman osteosarcoma cell line Saos-2 and the human embryonal kidney cellline HEK293 as performed in Example 2 (FIG. 7).

b) Performance in Vitro

Expression and Purification

For evaluation of the in vitro performance, the five superiorcombinations were expressed in the periplasm of E. coli (FIG. 6). Theamount of 0.1 l dYT-medium containing 35 mg/l chloramphenicol in shakingflasks was inoculated 1:40 with an over-night culture and incubated at30° C. Cells were induced at an OD550 of 1.5 with 1 mM IPTG andharvested after 2 hours of induction by centrifugation. For purificationof the scFvs, the cell pellet was resuspended and lysed by sonication.Following centrifugation in SS34 at 20 krpm, 4° C. for 30 minutes, thesupernatent was applied to a Ni-MC-affinity column (Hi-Trap™ ChelatingHP, 1 ml, Amersham Pharmacia) at pH 7.5 and eluted with 200 mM imidazolusing an Äkta Basic system from Amersham Pharmacia. The purity of thescFv fragments was greater than 98% as determined by SDS-PAGE (data notshown). The concentration of the purified protein was determined usingthe calculated extinction coefficient at 280 nm. The yield of solublepurified protein was normalized to a culture volume of 1 l with an OD600of 10 and varied from 8 to over 55 mg.

Resistance to Aggregation

Resistance towards aggregation has been shown to correlate withthermodynamic stability (Wörn, 1999) in vitro and the efficiency oftumor localization in a xenografted tumor model in mice (Willuda, 1999).In order to test for the stability, resistance to aggregation andreversibility of unfolding, 200 μl samples of the purified proteins atconcentrations of 6 μM in 50 mM Tris, pH 7.5, 100 mM NaCl were eitherkept 4 days at 4° C. or 4 days at 37° C. or 3 days at 4° C. followed byan incubation of 15 or 60 minutes at 100° C., slow cooling down to roomtemperature and an overnight incubation at 4° C. The oligomeric state ofeach sample was subsequently analyzed on a gel filtration columnequilibrated with 50 mM Tris, pH 7.5, 100 mM NaCl to estimate the amountof aggregated versus monomeric material (FIG. 8). The proteins wereinjected on a Superdex-75 column (Amersham Pharmacia) in a volume of 100μl and a flow-rate of 1 ml/min on a Äkta Basic system (AmershamPharmacia).

Resistance to Protease Degradation

To determine the stability of the isolated frameworks towards proteasedegradation, a parameter that is important for therapeutic applications,we incubated the purified frameworks in human serum at 37° C. (FIG. 9).

Purified, his-tagged scFv-protein (see above) at a concentration of 50μM was diluted tenfold into human serum to a final concentration of 5 μMin 90% serum. The samples were then either incubated at 37° C. foreither 3 days or 1 day, or taken directly for loading. Before loadinginsoluble and aggregated material was pelleted at maximal speed in aneppendorf centrifuge at 4° C. for 10 min. The supernatant was dilutedsix-fold with a loading dye to reduce the amount of serum loaded on thegel, heated to 100° C. for 5 min. and separated on a 12% SDS-PAGE. Thesoluble his-tagged scFv fragments were visualized by western blottingvia detection of the his-tag with an anti-his monoclonal mouse antibody(Qiagen) as primary and an anti-mouse-peroxidase conjugate (Sigma) assecondary antibody and using a chemoluminescent substrate (Pierce).SDS-PAGE and western blotting procedures are well known to a person ofordinary skill in the art.

EXAMPLE 6 Selection of Antigen Binders Through Screening of a RandomizedCDR-Library on the Framework 7.3 in the Interaction Screening System inYeast

Screening with the interaction system for antigen binders wasessentially performed as described in detail before (Auf der Maur,2002).

The plasmids for expression of the scFv-fusion constructs for screeningin yeast were derived from pESBA-Act2. It contains the yeast TRP1nutritional marker and the 2 micron origin of replication. Moreover ithas a constitutive actin promoter for strong expression and the GAL11transcriptional termination sequence, separated by a multiple cloningsite. For handling in bacterial systems, it also has a bacterial originof replication and the amp resistance gene.

The Gal4 activation domain (AD amino acids 768-881) was originallyamplified by PCR using pGAD424 (Clontech) as template with primersincluding the SV40 T-antigen nuclear localization signal N-terminal tothe Gal4-AD. The scFv library was obtained by PCR-amplification of thescFv-framework 7.3 using primers randomizing 7 amino acids within theCDR3 of VH. The resulting PCR-product was cloned in the framework 7.3,present in the vector in the orientation V_(L)-linker-V_(H), as aC-terminal fusion to Gal4-AD. Expression thus yields a fusion protein ofthe general structure Gal4-AD-scFv.

Screening was carried out in the yeast strain S. cerevisiae Immuna LHB(MATα ura3-52 leu2Δ1 trp1d63 his3Δ200 lys2Δ 385). It was derived fromthe strain JPY5 by integrating the divergently oriented LacZ and HIS3reporter genes under the control of a bi-directional promoter with sixLexA-binding sites (integrating reporter plasmid pDE200, Escher 2000)into the his3Δ200 locus and by integrating the LEU2 reporter gene underthe control of a promoter with eight LexA-binding sites (derived fromEGY48) into the leu2Δ1 locus.

Transcriptional activation of the reporter system is mediated by theGal4-AD moiety of the scFv-fusion construct, following the specificinteraction of its scFv moiety with the antigen-moiety of thebait-fusion protein. The bait-fusion protein consists of the kinasedomain of the human polo-like kinase 1 (hPlk1-KD) fused C-terminal tothe DNA-binding LexA protein. The kinase domain (amino acid 2-332) wasPCR amplified from a hPlk1 cDNA using the upstream primer5′-tgctctagaagt gctgcagtgactgcag-3′ (Seq. Id. No. 12) and downstreamprimer 5′-ggttgtcgacttacaggctgctgggagcaatcg-3′ (Seq. Id. No.13). Theresulting PCR product was cloned C-terminal of LexA via XbaI and SalIinto the bait vector. The bait vector contains the URA3 nutritionalmarker and an Ars Cen origin of replication. Expression of thebait-fusion protein is driven by a constitutively active actin promoter.Transcription is terminated by the GAL11 termination sequence. The baitvector also carries a bacterial origin of replication and the ampresistance gene for propagation in bacterial systems.

For screening the yeast strain S. cerevisiae Immuna LHB wasco-transformed with a scFv-library as fusion to Gal4-AD on thepESBA-Act2 vector and the bait-vector providing the LexA-hPLK1-KD fusionby following a standard lithium acetate transformation protocol (Agatep,1998). Following transformation, the cells were plated on drop-outplates (-Trp/-Leu/-Ura). Colonies were picked after 3 to 5 daysincubation at 30° C. and restreaked on drop-out plates (-Trp/-Leu/-Ura).Those that re-grew were tested for LacZ expression by development ofblue color in a filter assay on plates containing the substrate X-Gal.Positive clones were taken for further analysis involving isolation ofthe scFv-carrying plasmid from yeast, transformation into E. coli DH5α,isolation of plasmid from single colonies of E. coli, sequencing andre-transformation into freshly prepared yeast strain S. cerevisiaeImmuna LHB for the assay as described below. All methods were performedaccording to standard procedures, well known to a person of ordinaryskill in the art.

EXAMPLE 7 Evaluation of In Vivo Performance of Fab-Constructs Derivedfrom Novel scFv Frameworks

To evaluate the beneficial effect of using stable variable domainframeworks on different antibody formats, Fab expression vector wereconstructed for use in the yeast interaction screen.

a) Fab Constructs for Intracellular Screening in Yeast

Two different expression vectors were constructed to allow differentexpression levels. The vectors are based on either yEplac 112 (2 micron)or yCplac22 (ars/cen) backbones (Gietz, 1988). Both contain the yeastTRP1 nutritional marker, an inducible, bi-directional Gal1/Gal10promoter, a bacterial origin of replication and the amp resistance genefor handling in bacterial systems. In one direction, the VH domain ofthe framework 7.3 was cloned N-terminal to the CH1-domain of IgG1including the C-terminal cysteine, followed by a linker and the Gal4activation domain (AD amino acids 768-881) including the SV40 T-antigen.On the other side, the VL domain of the framework 7.3 was clonedN-terminal to the CL (lambda)-domain including the C-terminal cysteine.The terminators are Gal11 terminator on the side of the heavy chain andCyclin 1 terminator on the side of the light chain.

b) Performance in an Intracellular Assay in Yeast

For quantitative analysis of the performance of the antigen binders inscFv and Fab format in yeast (FIGS. 1 and 3), S. cerevisiae strainImmuna LHB was co-transformed with the isolated scFvs as Gal4-AD-fusionconstructs on the pESBA-Act2 vector and the bait vector containing theLexA-hPLK1-KD fusion by following a standard lithium acetatetransformation protocol (Agatep, 1998). Following transformation, thecells were plated on drop-out plates (-Trp, -Ura, Glc). 2 mlovernight-cultures in drop-out medium (-Trp, -Ura, Glc) were inoculatedin duplicates from streaks containing several colonies and grown at 30°C. Cultures were diluted in 1 ml drop-out medium (-Trp, -Ura, Gal) to anoptical density at 600 nm (OD600) of 0.7. They were grown at 30° C. for5 h. The assay was carried out as described above.

c) Expression of Soluble Protein Under Reducing Conditions in Yeast

To compare the yields of soluble protein upon expression under reducingconditions, the scFv and Fab constructs, together with the hPLK1-KD-baitvector, as described above were expressed in the cytoplasm of yeast S.cerevisiae. They were transformed as described above into the yeaststrain YDE173 and plated on -Trp, -Ura, drop-out plates containingglucose.

5 ml overnight-cultures in drop-out medium (Trp, -Ura, Glc) wereinoculated from streaks containing several colonies and grown at 30° C.Cultures were diluted in YPAG to an optical density at 600 nm (OD600) of0.5. They were grown at 30° C. for 7.5 h. For the native cell extract,2.5 ml cell culture normalized to an OD600 of 3 were harvested bycentrifugation, frozen in liquid nitrogen and subsequently resuspendedin 75 μl Y-PER (Pierce). The resuspended cell pellet was vortexedshortly and incubated slightly shaking at 20° C. for 20 min.Subsequently insoluble and aggregated material were pelleted at maximalspeed in an eppendorf centrifuge at 4° C. for 10 min. The supernatantwas mixed with loading dye, heated to 100° C. for 5 min and separated ona 12% SDS-PAGE. The soluble Gal4-AD-scFv fusion and the heavy chain partof the Fab fused to the Gal4-AD were visualized by western blotting viadetection of the Gal4-moiety with an anti-Gal4-AD monoclonal mouseantibody (Santa Cruz Biotechnology) as primary and ananti-mouse-peroxidase conjugate (Sigma) as secondary antibody and usinga chemoluminescent substrate (Pierce) (FIG. 11). SDS-PAGE and westernblotting procedures are well known to a person of ordinary skill in theart.

The invention claimed is:
 1. A single-chain antibody comprising avariable light chain framework VL and a variable heavy chain frameworkVH of a human and naturally occurring antibody which is highly stableand soluble under reducing conditions having the general structure:NH₂-VL-linker-VH-COOH; or NH₂-VH-linker-VL-COOH wherein the single-chainantibody has the VH framework and the VL framework of AJ, BJ, CJ, DJ,EJ, FJ, or GJ; wherein A is the amino acid sequence (Seq. Id. No. 1)EIVMTQSPSTLSASVGDRVIITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQYKSYWTFGQG TKLTVLG;

B is the amino acid sequence (Seq. Id. No. 2)EIVLTQSPSSLSASVGDRVTLTCRASQGIRNELAWYQQRPGKAPKRLIYAGSILQSGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQYYSLPYMFGQ GTKVDIKR;

C is the amino acid sequence (Seq. Id. No. 3)EIVMTQSPATLSVSPGESAALSCRASQGVSTNVAWYQQKPGQAPRLLIYGATTRASGVPARFSGSGSGTEFTLTINSLQSEDFAAYYCQQYKHWPPWTFG QGTKVEIKR;

D is the amino acid sequence (Seq. Id. No. 4)QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQLPGTAPQLLIYDNTKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSGVV FGGGTKILTVLG;

E is the amino acid sequence (Seq. Id. No. 5)EIVLTQSPATLSLSPGERATLSCRASQTLTHYLAWYQQKPGQAPRLLTYDTSKRATGVPARFSGSGSGTDFTLTISSLEPEDSALYYCQQRNSWPHTFGG GTKLEIKR;

F is the amino acid sequence (Seq. Id. No. 6)SYVLTQPPSVSVAPGQTATVTCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTIRRVEAGDEADYYCQVWDSSSDHNVFG SGTKVEIKR;

G is the amino acid sequence (Seq. Id. No. 7)LPVLTQPPSVSVAPGQTARISCGGNNIETISVHWYQQKPGQAPVLVVSDDSVRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDYVVFG GGTKLTVLG;

H is the amino acid sequence (Seq. Id. No. 8)QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAHVLRFLEWLPDAFDIWGQGTLVTVSS;

I is the amino acid sequence (Seq. Id. No. 9)EIVLTQSPSSLSASLGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSSQSGVPSRFRGSESGTDFTLTISNLQPEDFATYYCQQSYRTPFTFGP GTKVETKR; and

J is the amino acid sequence (Seq. Id. No. 10)VQLVQSGAEVKKPGASVKVSCTASGYSFTGYFLHWVRQAPGQGLEWMGRINPDSGDTIYAQKFQDRVTLTRDTSIGTVYMELTSLTSDDTAVYYCARVPRGTYLDPWDYFDYWGQGTLVTVSS.


2. A single-chain framework fused to a second protein moiety to yield afusion construct of the general structure: NH₂-VL-linker-VH-secondprotein-COOH; or NH₂-second protein-VL-linker-VH-COOH wherein thesingle-chain framework has the VH framework and the VL framework of AJ,BJ, CJ, DJ, EJ, FJ, or GJ; wherein A is the amino acid sequence (Seq.Id. No. 1) EIVMTQSPSTLSASVGDRVIITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQYKSYWTFGQG TKLTVLG;

B is the amino acid sequence (Seq. Id. No. 2)EIVLTQSPSSLSASVGDRVTLTCRASQGIRNELAWYQQRPGKAPKRLIYAGSILQSGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQYYSLPYMFGQ GTKVDIKR;

C is the amino acid sequence (Seq. Id. No. 3)EIVMTQSPATLSVSPGESAALSCRASQGVSTNVAWYQQKPGQAPRLLIYGATTRASGVPARFSGSGSGTEFTLTINSLQSEDFAAYYCQQYKHWPPWTFG QGTKVEIKR;

D is the amino acid sequence (Seq. Id. No. 4)QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQLPGTAPQLLIYDNTKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSGVV FGGGTKLTVLG;

E is the amino acid sequence (Seq. Id. No. 5)EIVLTQSPATLSLSPGERATLSCRASQTLTHYLAWYQQKPGQAPRLLIYDTSKRATGVPARFSGSGSGTDFTLTISSLEPEDSALYYCQQRNSWPHTFGG GTKLEIKR;

F is the amino acid sequence (Seq. Id. No. 6)SYVLTQPPSVSVAPGQTATVTCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTIRRVEAGDEADYYCQVWDSSSDHNVFG SGTKVEIKR;

G is the amino acid sequence (Seq. Id. No. 7)LPVLTQPPSVSVAPGQTARISCGGNNIETISVHWYQQKPGQAPVLVVSDDSVRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDYVVFG GGTKLTVLG;

H is the amino acid sequence (Seq. Id. No. 8)QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAHVLRFLEWLPDAFDIWGQGTLVTVSS;

I is the amino acid sequence (Seq. Id. No. 9)EIVLTQSPSSLSASLGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSSQSGVPSRFRGSESGTDFTLTISNLQPEDFATYYCQQSYRTPFTFGP GTKVEIKR; and

J is the amino acid sequence (Seq. Id. No. 10)VQLVQSGAEVKKPGASVKVSCTASGYSFTGYFLHWVRQAPGQGLEWMGRINPDSGDTIYAQKFQDRVTLTRDTSIGTVYMELTSLTSDDTAVYYCARVPRGTYLDPWDYFDYWGQGTLVTVSS.


3. The single chain antibody of claim 1, wherein orientation of the VHand VL regions is reversed.
 4. The single chain antibody according toclaim 1 wherein the VL framework is of the kappa1, lambda 1 or 3 type.5. The single chain framework according to claim 2, wherein the secondprotein provides a read-out for intracellular assays.
 6. A single chainantibody selected from the group consisting of variants of the singlechain frameworks according to claim 1 or
 2. 7. A single chain antibodyselected from the group consisting of derivatives of the single chainframeworks according to claim 1 or
 2. 8. An antibody comprising the VLor the VH or both from the single chain framework according to claim 1.9. An antibody fragment comprising at least one variable domain of animmunoglobulin and comprising the VL or the VH or both from the singlechain framework according to claim 1.